- Email: [email protected]
Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway
Accepted Manuscript Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway Wichida Chaweewannakorn, Wataru Ariyoshi, Toshinori Okinaga, Kazumasa Morikawa, Katsura Saeki, Kenshi Maki, Tatsuji Nishihara PII:
S0006-291X(17)30256-5
DOI:
10.1016/j.bbrc.2017.01.181
Reference:
YBBRC 37243
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 21 January 2017 Accepted Date: 31 January 2017
Please cite this article as: W. Chaweewannakorn, W. Ariyoshi, T. Okinaga, K. Morikawa, K. Saeki, K. Maki, T. Nishihara, Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.181. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ameloblastin and Enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway
Wichida Chaweewannakorna,b, Wataru Ariyoshia, Toshinori Okinagaa, Kazumasa
a
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Morikawab, Katsura Saekib, Kenshi Makib, Tatsuji Nishiharaa,* Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu
Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka, 803-8580,
b
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Japan
Division of Developmental Stomatognathic Function Science, Department of Health
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Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka, 803-8580, Japan
*Corresponding author: Tatsuji Nishihara
Fax: (+81)93 581 4984
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Tel: (+81)93 285 3051
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products
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also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-
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dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on
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osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover,
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pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam
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downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may
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indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.
Keywords
Enamel-related gene products, Ameloblastin, Enamelin,
Abbreviations RANKL: receptor activator of nuclear factor kappa-B ligand; 1,25(OH)2D3: 1,25-dihydroxy2
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vitamin D3; DEX: dexamethasone; MAPK: mitogen-activated protein kinase; ERK1/2: extracellular-signal-regulated kinases1/2; JNK: Jun amino-terminal kinases; OPG: osteoprotegerin; α-MEM: Minimum Essential Medium Eagle Alpha Modification; BMCs: bone marrow-derived macrophages; TRAP: tartrate-resistant acid phosphatase
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Introduction Bone remodeling involves the synthesis and resorption of bone microstructure. The mechanisms that regulate bone remodeling are highly complex and involve several cell types, growth factors, and cytokines [1]. Bone remodeling begins with the development of
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osteoblasts from mesenchymal progenitor cells, while osteoclasts differentiate from
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hematopoietic precursor cells. Mature osteoblasts express the receptor activator of nuclear factor kappa-B ligand (RANKL), which is a member of the tumor necrosis factor (TNF) superfamily. This cytokine works in coordination with other factors, leading to multinucleated osteoclast differentiation and bone resorption [2-4]. Osteoprotegerin (OPG), a decoy receptor of RANKL, is also generated by osteoblasts and supporting stromal cells, and
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has been shown to block RANK-RANKL coupling [5]. RANKL is a type II transmembrane protein that can be found in both soluble and
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membrane-bound forms. RANKL is synthesized in response to both systemic and local factors, including osteotropic hormones 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and
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glucocorticoid dexamethasone (DEX) [6,7]. Previous studies suggest that it is not only boneforming osteoblasts that can express RANKL, but also bone supporting stromal cells, osteocytes, and T-lymphocytes [6,8,9]. Ameloblastin (Ambn) and Enamelin (Enam), a member of enamel-related gene
products (ERPs), are extracellular matrix proteins. Ambn and Enam are encoded by a cluster of secretory calcium-binding phosphoprotein-encoded genes, which include small integrinbinding ligand, N-linked glycoprotein (SIBLING) proteins, milk, and saliva proteins [10]. Although ERPs are primarily detected in enamel during tooth development, recent studies 3
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have revealed that it can be found in dentin, pulp, cementum, the periodontal ligament, craniofacial bones, and long bones, suggesting that ERP functions are not restricted to amelogenesis; indeed, they appear to play a role in the development and differentiation of other mineralized tissues [11-13]. Previous studies have been reported regarding the role of
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Ambn in osteogenic differentiation, cranial suture closure, long bone growth and mineralization, and bone fracture healing [14-17]. Moreover, Enam has been found to play a role in bone metabolism [18]. However, the mechanism by which Ambn or Enam affect
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osteoclastogenesis has not been fully elucidated.
In the present study, we focused on the biological effects of Ambn and Enam on
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osteoclast-supporting cells, and attempted to propose the underlying mechanisms involved in the process. To clarify the biological mechanism, we created an in vitro assay system using ST2 cells, and found that Ambn and Enam suppress osteoclast formation via downregulation
Materials and Methods Reagents and antibodies
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of RANKL.
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Recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) were obtained from Abnova Corp. (Taipei, Taiwan). Recombinant human soluble RANKL
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(rhRANKL) and recombinant human macrophage colony stimulating factor (rhM-CSF) were purchased from Peprotech (Rocky Hill, NJ, USA). Phospho-p38 MAPK monoclonal antibody, p38 MAPK polyclonal antibody, phospho-JNK polyclonal antibody, JNK polyclonal antibody, phospho-ERK1/2 polyclonal antibody, and ERK1/2 monoclonal antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-βactin monoclonal antibody, 1,25(OH)2D3, DEX, and IgG from mouse serum were purchased from Sigma–Aldrich (St. Louis, MO, USA). A mouse TRANCE/TNFSF11/RANKL antibody
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was purchased from R&D Systems (Minneapolis, MN, USA). U0126, SB203580, and JNK inhibitor II were obtained from Merck Millipore (Billerica, MA, USA). Cell cultures ST2 cells were obtained from the Riken Cell Bank and maintained in Minimum
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Essential Medium Eagle Alpha Modification (α-MEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), penicillin G (66 µg/ml) (Nacalai Tesque, Kyoto, Japan), and streptomycin (140 µg/ml) (Wako Pure Chemical Industries, Osaka, Japan) at 37°C in a humidified atmosphere with 5% CO2. After reaching
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the confluence stage, cells were trypsinized and replated at a concentration of 5×105
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cells/well and incubated in α-MEM with 10% FBS overnight. The ST2 cells were induced with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for indicated periods of time. To identify the effects of Ambn or Enam on RANKL and OPG expression, cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, followed by stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. In some experiments, ST2 cells were pretreated with 10 µM
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U0126, 10 µM SB203580, or 10 µM JNK inhibitor II for 1 hour before stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX in order to observe the influence of MAPK on RANKL
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expression.
Reverse transcriptase-qualitative polymerase chain reaction (RT-qPCR)
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Total mRNA in ST2 cells was extracted by Cica Geneus® PCR prep kit (Kanto Chemical Co., Inc., Tokyo, Japan) per manufacturer instructions. The RNA was reversetranscribed with ReverTra Ace qPCR RT Master Mix (Toyobo Co., LTD, Osaka, Japan). PCR products were detected using FAST SYBR® Green Master Mix (Applied Biosystems, Foster City, CA) with following specific primer sequences: β-actin, 5’-GCTGTGCTATG TTGCTCTAGACTT-3’
(forward)
and
5’-AATTGAATGTAGTTTCATGGATGC-3’
(reverse); RANKL, 5’-GGCCAC AGCGCTTCTCA-3’ (forward) and 5’-CCTCGCTGG
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GCCACATC-3’ (reverse); OPG, 5’-GCC TGGGACCAAAGTGAATG-3’ (forward) and 5’CTTGTGAGCTGTGTCTCCGTTT-3’ (reverse). Thermal cycling and fluorescence detection were performed using a StepOnTM Real-Time PCR System (Applied Biosystems). The samples were normalized by using primers specific to β-actin, and relative expression levels
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were calculated by the 2-∆∆Ct method. Western blot analysis
Whole cell lysates were extracted using Cell Lysis Buffer (Cell Signaling Technology
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Inc., USA) containing a protease inhibitor (Thermo Scientific, Rockford, IL) and phosphatase inhibitor mixture (Nacalai Tesque). Total protein concentration was measured by a DC
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protein assay kit (Bio-Rad, Hercules, CA, USA). Equivalent amounts of protein were subjected to 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Merck Millipore). The membrane non-specific binding sites were blocked with Blocking One (Nacalai Tesque) for 1 hour at room temperature, and then incubated with diluted primary antibodies against RANKL, ERK1/2,
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phospho-ERK1/2, p38 MAPK, phospho-p38 MAPK, JNK, phospho-JNK, and β-actin at 4°C overnight. This was followed by incubation with secondary antibodies conjugated to
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horseradish peroxidase (GE Healthcare, Little Chalfont, UK) for 1 hour at room temperature. For signal detection, chemiluminescence was produced using ECL reagent (GE Healthcare)
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and detected digitally with a GelDocTM XR Plus system (Bio-Rad). Mouse bone marrow cell isolation Bone marrow cells were extracted from 6-week old ddY male mice from Kiwa
Experimental Animal Laboratory, Wakayama, Japan. Tibias and femurs of ddY male mice were dissected, and scissors cut off the ends. The bone marrow cavity was flushed with αMEM medium via 25-gauge needles, and the cell suspension was centrifuged at 1200 rpm at 4°C for 5 minutes. The supernatant was aspirated, and pellets were resuspended in α-MEM with 10% FBS and 20 ng/ml rhM-CSF. Cells were seeded in 10-cm plates and cultured for 3 6
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days at 37°C and 5% CO2 in a humid atmosphere. Adherent cells were defined as bone marrow-derived macrophages (BMCs). Co-culture system ST2 cells were plated at a concentration of 3×103 cells/well in 48-well plates and
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maintained in α-MEM containing 10% FBS, 10-7 M 1,25(OH)2D3, and 10-7 M DEX overnight. In some experiments, ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam 6 hours prior to stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. To confirm
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the effects of RANKL on multinucleated osteoclast formation, ST2 cells were blocked with 60–500 ng/ml anti-TRANCE/TNFSF11/RANKL or mouse IgG for 2 hours, and subsequently
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stimulated with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. BMCs (1×105 cells/well) were added to prepared ST2 cells in 48-well plates. The cell co-culture system was incubated in α-MEM with 10% FBS in the presence of 10-7 M 1,25(OH)2D3 and 10-7 M DEX, with a medium change every 2 days. On day 4, cells were subjected to tartrate-resistant acid phosphatase (TRAP) analysis.
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TRAP analysis
Adherent cells were fixed and stained with Leukocyte Acid Phosphatase Kit (Sigma-
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Aldrich) per manufacturer protocol. TRAP-positive multinucleated cells containing more than three nuclei were defined as osteoclasts.
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Statistical analysis
All data were obtained from three independent experiments, and each experiment was
performed in triplicate. Statistical differences were determined using one-way analysis of variance followed by Tukey’s post hoc test. A p-value<0.05 was considered statistically significant.
Results
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Ambn and Enam suppressed RANKL expression on ST2 cells induced with 1,25(OH)2D3 and DEX We first confirmed RANKL and OPG expression in ST2 cells induced with 1,25(OH)2D3 and DEX, and found that RANKL gene and protein expression were increased
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for 24 hours, while OPG was reduced (data not shown). To further verify the effect of Ambn and Enam on RANKL and OPG expression, we pretreated ST2 cells with rhAmbn or rhEnam prior to stimulation with 1,25(OH)2D3 and DEX. Real-time RT-qPCR revealed that the
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RANKL and OPG gene expression regulated by 1,25(OH)2D3 and DEX were suppressed by both Ambn and Enam pretreatment (Figs. 1A, B). Western blot analysis also showed that
cells (Fig. 1C).
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stimulation with rhAmbn or rhEnam resulted in decreased RANKL protein expression in ST2
Ambn and Enam downregulated RANKL expression on ST2 cells via p38 MAPK and ERK1/2 signaling pathways
To further understand the mechanisms by which Ambn and Enam attenuated the
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expression of RANKL on ST2 cells stimulated with 1,25(OH)2D3 and DEX, we concentrated on the MAPK signaling pathways. As shown in Figure 2A, 1,25(OH)2D3 and DEX
120 minutes.
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transiently phosphorylated ERK1/2, p38MAPK, and JNK proteins in ST2 cells from 15 to
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To examine pivotal roles of Ambn and Enam on MAPK signaling pathways, ST2 cells were pretreated with rhAmbn or rhEnam at the indicated times, followed by stimulation with 1,25(OH)2D3 and DEX. Pretreatment with Ambn and Enam significantly reduced phosphorylation of p38 MAPK and JNK at 15 minutes. In addition, the phosphorylation of all MAPK was decreased at 30 minutes (Fig. 2B). Moreover, to address the effect of MAPK on RANKL expression, we incubated ST2 cells with MAPK inhibitors U0126 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), or JNK inhibitor II (JNK inhibitor) before treatment with 1,25(OH)2D3 and DEX. Both real-time RT8
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qPCR and western blot analysis demonstrated that U0126 and SB203580 suppressed RANKL expression induced with 1,25(OH)2D3 and DEX. In contrast, JNK inhibitor II enhanced the expression of RANKL insignificantly (Figs. 2C, D). Ambn and Enam inhibited osteoclast differentiation in co-culture system between ST2
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cells and BMCs To determine whether Ambn or Enam play a role in ST2 cell-supported osteoclast formation, ST2 cells were pretreated with rhAmbn or rhEnam prior to stimulation with
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1,25(OH)2D3 and DEX, and then co-cultured with BMCs. After 4 days, several TRAPpositive multinucleated osteoclasts were identified. We found that the differentiation of
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BMCs into multinucleated osteoclasts induced with 1,25(OH)2D3 and DEX was clearly decreased under the preincubation of ST2 cells in the presence of Ambn or Enam (Fig. 3). These results indicate that Ambn and Enam adversely affect osteoclastogenesis, suggesting that these proteins directly affect ST2 cells.
differentiation
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RANKL is an essential factor for multinucleated osteoclast formation and
To further examine the critical roles of RANKL on multinucleated osteoclast
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formation, we blocked ST2 cells with various concentrations of neutralizing antibody against RANKL (60–500 ng/ml), followed by induction with 1,25(OH)2D3 and DEX, and then co-
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culture with BMCs. The neutralizing antibody-treated groups obviously displayed lower numbers of multinucleated osteoclasts compared with mouse-IgG control groups, in a dosedependent manner (Fig. 4).
Discussion In the present study, we aimed to identify the role of Ambn and Enam on RANKL expression, and to verify the effects of Ambn and Enam on osteoclastogenesis. Our data revealed that exogenous Ambn and Enam suppressed RANKL expression, which attenuated 9
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multinucleated osteoclast formation. This finding is consistent with previous reports suggesting that Ambn-deficient mice exhibited lower bone volume density and higher trabecular bone separation in femurs [16,17]. Moreover, Ambn was found to promote bone mineralization by binding to CD63, and then inducing Src kinase inhibition in an
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osteosarcoma cell line [19]. In contrast, coating recombinant Ambn on cell culture plates increased the number of the multinucleated osteoclast formation by the integrin/ERK pathway [20], suggesting that Ambn has both direct and indirect effects on osteoclast
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formation. We do not have a specific explanation for these contrasting results, but it is possible that they reflect the differences in culture conditions, types of cells, concentrations
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of Ambn, and protocols used in the studies.
We also observed the enhancement of endogenous ERP gene expression in ST2 cells induced with 1,25(OH)2D3 and DEX, which was consistent with key time point marker genes of osteoblast differentiation (data not shown). However, the previous study demonstrated that commitment to osteoblast lineage was not mandatory for fibroblastic cell types to express
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RANKL, and mature osteoblasts are not a significant source of RANKL in bone [21]. Thus, in our study, ST2 cells serve as a supporting element for generating multinucleated
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osteoclasts from BMCs by producing RANKL with stimulation by 1,25(OH)2D3 and DEX. 1,25(OH)2D3 is one of the essential osteotropic factors that has been firmly reported to
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induce the differentiation of osteoclast progenitors cells in both direct and indirect pathways. 1,25(OH)2D3 itself has been found to promote multinucleated osteoclast formation [22]. Furthermore, 1,25(OH)2D3 indirectly supports osteoclastogenesis by stimulating the expression of RANKL in osteoblasts, and stromal cells pass through genomic and nongenomic pathways [23]. It has been found to activate RANKL gene transcription through vitamin D-responsive elements (VDRE) [24,25]. Additionally, DEX has been shown to enhance osteoclast formation through various mechanisms, including elevating VDR transcription and inducing the expression of M-CSF in ST2 and MC3T3E1 cells [26,27]. In 10
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this experiment, we could not detect the relevance of Ambn and Enam on nuclear translocation of VDR (Data not shown), but we found that Ambn and Enam decreased the phosphorylation of all MAPK induced with 1,25(OH)2D3 and DEX. However, only specific inhibitors against ERK1/2 and p38MAPK displayed an ability to contribute to RANKL
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expression in ST2 cells. MAPK proteins are known to play an essential role in controlling cell proliferation, differentiation, and gene expression. In mammals, MAPK can be divided into three main
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families: ERK1/2, JNK and p38 MAPK [28,29]. MAPK-specific inhibitors proved that activation of ERK and p38, specific MAPK proteins, was imperative for triggering RANKL
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expression in bone marrow stromal cells and osteoblasts [30]. Other studies have also supported the function of ERK1/2 and p38 in RANKL expression [30,31]. In our study, compared with p38, ERK1/2 has only minor effects on RANKL expression. From these results, the inhibitory effect of Ambn and Enam on the expression of RANKL induced with 1,25(OH)2D3 and DEX was found to depend primarily on the regulation of the p38 MAPK
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pathway.
In conclusion, our study demonstrated that Ambn and Enam might attenuate
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osteoclastogenesis by suppressing RANKL expression via MAPK signaling pathways in stromal cells. Further research is needed to precisely elucidate the molecular mechanism of
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ERPs on bone remodeling, which may impact therapies for bone metabolic diseases in the future.
Conflicts of Interest The authors have no financial conflicts of interest to disclose.
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Funding
This work was partially supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science [grant numbers 26463115]
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Figure Legends
Figure 1. Ambn and Enam suppressed RANKL expression on ST2 cells induced with 1,25(OH)2D3 and DEX. ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, then stimulated with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 24 hours
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(real-time RT-qPCR) or 36 hours (western blot analysis). (A) The mRNA level of RANKL and OPG was measured by real-time RT-qPCR. Data are expressed as the mean ± S.D. of triplicate cultures. *p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups. (B)
for RANKL. β-actin served as an internal control.
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Whole cell lysates were subjected to SDS-PAGE and western blot analysis with blot probes
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Figure 2. Ambn and Enam downregulated RANKL expression on ST2 cells via p38 MAPK and ERK1/2 signaling pathways. (A) ST2 cells were treated with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 15–180 minutes. (B) ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, prior to stimulation with 10-7 M
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1,25(OH)2D3 and 10-7 M DEX for 15 and 30 minutes. Whole cell lysates were subjected to western blot analysis for detecting MAPK expression and phosphorylation. (C, D) ST2 cells were blocked with 10 µM U0126, JNK inhibitor II or SB203580 for 1 hour followed by
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stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 24 (C) or 36 (D) hours. (C) The mRNA level of RANKL was measured by real-time RT-qPCR. Data are expressed as the
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mean ± S.D. of triplicate cultures. *p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups. (D) Whole cell lysates were subjected to western blot analysis with the blot probes for RANKL.
Figure 3. Ambn and Enam inhibit osteoclast differentiation in the co-culture system between ST2 cells and BMCs. (A) ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, prior to stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. After 24 hours, ST2 cells were co-cultured with BMCs for 4 days. TRAP analysis was performed to detect multinucleated osteoclast formation. Scale bar 500 µm. (B) TRAP16
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positive cells containing more than three nuclei were counted. Data are expressed as the mean ± S.D. of triplicate cultures. *p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups. Figure 4. RANKL is essential for generating multinucleated osteoclasts. (A) ST2 cells were cultured in a medium containing 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 22 hours,
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then blocked with 60–500 ng/ml anti-RANKL antibody or mouse IgG, followed by coculture with BMCs for 4 days. Scale bar 500 µm. (B) Several TRAP-positive multinucleated osteoclasts were counted. Data are expressed as the mean ± S.D. of triplicate cultures.
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ACCEPTED MANUSCRIPT Highlights •
Ambn and Enam affect RANKL expression through MAPK signaling pathway
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MAPK signaling pathway is necessary for RANKL expression in bone marrow stromal
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Ambn and Enam indirectly regulate multinucleated osteoclast formation.
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cell.
S0006-291X(17)30256-5
DOI:
10.1016/j.bbrc.2017.01.181
Reference:
YBBRC 37243
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 21 January 2017 Accepted Date: 31 January 2017
Please cite this article as: W. Chaweewannakorn, W. Ariyoshi, T. Okinaga, K. Morikawa, K. Saeki, K. Maki, T. Nishihara, Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.181. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ameloblastin and Enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway
Wichida Chaweewannakorna,b, Wataru Ariyoshia, Toshinori Okinagaa, Kazumasa
a
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Morikawab, Katsura Saekib, Kenshi Makib, Tatsuji Nishiharaa,* Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu
Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka, 803-8580,
b
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Japan
Division of Developmental Stomatognathic Function Science, Department of Health
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Promotion, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka, 803-8580, Japan
*Corresponding author: Tatsuji Nishihara
Fax: (+81)93 581 4984
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Tel: (+81)93 285 3051
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products
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also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-
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dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on
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osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover,
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pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam
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downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may
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indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.
Keywords
Enamel-related gene products, Ameloblastin, Enamelin,
Abbreviations RANKL: receptor activator of nuclear factor kappa-B ligand; 1,25(OH)2D3: 1,25-dihydroxy2
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vitamin D3; DEX: dexamethasone; MAPK: mitogen-activated protein kinase; ERK1/2: extracellular-signal-regulated kinases1/2; JNK: Jun amino-terminal kinases; OPG: osteoprotegerin; α-MEM: Minimum Essential Medium Eagle Alpha Modification; BMCs: bone marrow-derived macrophages; TRAP: tartrate-resistant acid phosphatase
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Introduction Bone remodeling involves the synthesis and resorption of bone microstructure. The mechanisms that regulate bone remodeling are highly complex and involve several cell types, growth factors, and cytokines [1]. Bone remodeling begins with the development of
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osteoblasts from mesenchymal progenitor cells, while osteoclasts differentiate from
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hematopoietic precursor cells. Mature osteoblasts express the receptor activator of nuclear factor kappa-B ligand (RANKL), which is a member of the tumor necrosis factor (TNF) superfamily. This cytokine works in coordination with other factors, leading to multinucleated osteoclast differentiation and bone resorption [2-4]. Osteoprotegerin (OPG), a decoy receptor of RANKL, is also generated by osteoblasts and supporting stromal cells, and
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has been shown to block RANK-RANKL coupling [5]. RANKL is a type II transmembrane protein that can be found in both soluble and
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membrane-bound forms. RANKL is synthesized in response to both systemic and local factors, including osteotropic hormones 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and
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glucocorticoid dexamethasone (DEX) [6,7]. Previous studies suggest that it is not only boneforming osteoblasts that can express RANKL, but also bone supporting stromal cells, osteocytes, and T-lymphocytes [6,8,9]. Ameloblastin (Ambn) and Enamelin (Enam), a member of enamel-related gene
products (ERPs), are extracellular matrix proteins. Ambn and Enam are encoded by a cluster of secretory calcium-binding phosphoprotein-encoded genes, which include small integrinbinding ligand, N-linked glycoprotein (SIBLING) proteins, milk, and saliva proteins [10]. Although ERPs are primarily detected in enamel during tooth development, recent studies 3
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have revealed that it can be found in dentin, pulp, cementum, the periodontal ligament, craniofacial bones, and long bones, suggesting that ERP functions are not restricted to amelogenesis; indeed, they appear to play a role in the development and differentiation of other mineralized tissues [11-13]. Previous studies have been reported regarding the role of
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Ambn in osteogenic differentiation, cranial suture closure, long bone growth and mineralization, and bone fracture healing [14-17]. Moreover, Enam has been found to play a role in bone metabolism [18]. However, the mechanism by which Ambn or Enam affect
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osteoclastogenesis has not been fully elucidated.
In the present study, we focused on the biological effects of Ambn and Enam on
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osteoclast-supporting cells, and attempted to propose the underlying mechanisms involved in the process. To clarify the biological mechanism, we created an in vitro assay system using ST2 cells, and found that Ambn and Enam suppress osteoclast formation via downregulation
Materials and Methods Reagents and antibodies
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of RANKL.
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Recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) were obtained from Abnova Corp. (Taipei, Taiwan). Recombinant human soluble RANKL
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(rhRANKL) and recombinant human macrophage colony stimulating factor (rhM-CSF) were purchased from Peprotech (Rocky Hill, NJ, USA). Phospho-p38 MAPK monoclonal antibody, p38 MAPK polyclonal antibody, phospho-JNK polyclonal antibody, JNK polyclonal antibody, phospho-ERK1/2 polyclonal antibody, and ERK1/2 monoclonal antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-βactin monoclonal antibody, 1,25(OH)2D3, DEX, and IgG from mouse serum were purchased from Sigma–Aldrich (St. Louis, MO, USA). A mouse TRANCE/TNFSF11/RANKL antibody
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was purchased from R&D Systems (Minneapolis, MN, USA). U0126, SB203580, and JNK inhibitor II were obtained from Merck Millipore (Billerica, MA, USA). Cell cultures ST2 cells were obtained from the Riken Cell Bank and maintained in Minimum
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Essential Medium Eagle Alpha Modification (α-MEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), penicillin G (66 µg/ml) (Nacalai Tesque, Kyoto, Japan), and streptomycin (140 µg/ml) (Wako Pure Chemical Industries, Osaka, Japan) at 37°C in a humidified atmosphere with 5% CO2. After reaching
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the confluence stage, cells were trypsinized and replated at a concentration of 5×105
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cells/well and incubated in α-MEM with 10% FBS overnight. The ST2 cells were induced with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for indicated periods of time. To identify the effects of Ambn or Enam on RANKL and OPG expression, cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, followed by stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. In some experiments, ST2 cells were pretreated with 10 µM
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U0126, 10 µM SB203580, or 10 µM JNK inhibitor II for 1 hour before stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX in order to observe the influence of MAPK on RANKL
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expression.
Reverse transcriptase-qualitative polymerase chain reaction (RT-qPCR)
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Total mRNA in ST2 cells was extracted by Cica Geneus® PCR prep kit (Kanto Chemical Co., Inc., Tokyo, Japan) per manufacturer instructions. The RNA was reversetranscribed with ReverTra Ace qPCR RT Master Mix (Toyobo Co., LTD, Osaka, Japan). PCR products were detected using FAST SYBR® Green Master Mix (Applied Biosystems, Foster City, CA) with following specific primer sequences: β-actin, 5’-GCTGTGCTATG TTGCTCTAGACTT-3’
(forward)
and
5’-AATTGAATGTAGTTTCATGGATGC-3’
(reverse); RANKL, 5’-GGCCAC AGCGCTTCTCA-3’ (forward) and 5’-CCTCGCTGG
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GCCACATC-3’ (reverse); OPG, 5’-GCC TGGGACCAAAGTGAATG-3’ (forward) and 5’CTTGTGAGCTGTGTCTCCGTTT-3’ (reverse). Thermal cycling and fluorescence detection were performed using a StepOnTM Real-Time PCR System (Applied Biosystems). The samples were normalized by using primers specific to β-actin, and relative expression levels
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were calculated by the 2-∆∆Ct method. Western blot analysis
Whole cell lysates were extracted using Cell Lysis Buffer (Cell Signaling Technology
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Inc., USA) containing a protease inhibitor (Thermo Scientific, Rockford, IL) and phosphatase inhibitor mixture (Nacalai Tesque). Total protein concentration was measured by a DC
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protein assay kit (Bio-Rad, Hercules, CA, USA). Equivalent amounts of protein were subjected to 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Merck Millipore). The membrane non-specific binding sites were blocked with Blocking One (Nacalai Tesque) for 1 hour at room temperature, and then incubated with diluted primary antibodies against RANKL, ERK1/2,
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phospho-ERK1/2, p38 MAPK, phospho-p38 MAPK, JNK, phospho-JNK, and β-actin at 4°C overnight. This was followed by incubation with secondary antibodies conjugated to
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horseradish peroxidase (GE Healthcare, Little Chalfont, UK) for 1 hour at room temperature. For signal detection, chemiluminescence was produced using ECL reagent (GE Healthcare)
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and detected digitally with a GelDocTM XR Plus system (Bio-Rad). Mouse bone marrow cell isolation Bone marrow cells were extracted from 6-week old ddY male mice from Kiwa
Experimental Animal Laboratory, Wakayama, Japan. Tibias and femurs of ddY male mice were dissected, and scissors cut off the ends. The bone marrow cavity was flushed with αMEM medium via 25-gauge needles, and the cell suspension was centrifuged at 1200 rpm at 4°C for 5 minutes. The supernatant was aspirated, and pellets were resuspended in α-MEM with 10% FBS and 20 ng/ml rhM-CSF. Cells were seeded in 10-cm plates and cultured for 3 6
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days at 37°C and 5% CO2 in a humid atmosphere. Adherent cells were defined as bone marrow-derived macrophages (BMCs). Co-culture system ST2 cells were plated at a concentration of 3×103 cells/well in 48-well plates and
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maintained in α-MEM containing 10% FBS, 10-7 M 1,25(OH)2D3, and 10-7 M DEX overnight. In some experiments, ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam 6 hours prior to stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. To confirm
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the effects of RANKL on multinucleated osteoclast formation, ST2 cells were blocked with 60–500 ng/ml anti-TRANCE/TNFSF11/RANKL or mouse IgG for 2 hours, and subsequently
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stimulated with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. BMCs (1×105 cells/well) were added to prepared ST2 cells in 48-well plates. The cell co-culture system was incubated in α-MEM with 10% FBS in the presence of 10-7 M 1,25(OH)2D3 and 10-7 M DEX, with a medium change every 2 days. On day 4, cells were subjected to tartrate-resistant acid phosphatase (TRAP) analysis.
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TRAP analysis
Adherent cells were fixed and stained with Leukocyte Acid Phosphatase Kit (Sigma-
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Aldrich) per manufacturer protocol. TRAP-positive multinucleated cells containing more than three nuclei were defined as osteoclasts.
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Statistical analysis
All data were obtained from three independent experiments, and each experiment was
performed in triplicate. Statistical differences were determined using one-way analysis of variance followed by Tukey’s post hoc test. A p-value<0.05 was considered statistically significant.
Results
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Ambn and Enam suppressed RANKL expression on ST2 cells induced with 1,25(OH)2D3 and DEX We first confirmed RANKL and OPG expression in ST2 cells induced with 1,25(OH)2D3 and DEX, and found that RANKL gene and protein expression were increased
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for 24 hours, while OPG was reduced (data not shown). To further verify the effect of Ambn and Enam on RANKL and OPG expression, we pretreated ST2 cells with rhAmbn or rhEnam prior to stimulation with 1,25(OH)2D3 and DEX. Real-time RT-qPCR revealed that the
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RANKL and OPG gene expression regulated by 1,25(OH)2D3 and DEX were suppressed by both Ambn and Enam pretreatment (Figs. 1A, B). Western blot analysis also showed that
cells (Fig. 1C).
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stimulation with rhAmbn or rhEnam resulted in decreased RANKL protein expression in ST2
Ambn and Enam downregulated RANKL expression on ST2 cells via p38 MAPK and ERK1/2 signaling pathways
To further understand the mechanisms by which Ambn and Enam attenuated the
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expression of RANKL on ST2 cells stimulated with 1,25(OH)2D3 and DEX, we concentrated on the MAPK signaling pathways. As shown in Figure 2A, 1,25(OH)2D3 and DEX
120 minutes.
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transiently phosphorylated ERK1/2, p38MAPK, and JNK proteins in ST2 cells from 15 to
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To examine pivotal roles of Ambn and Enam on MAPK signaling pathways, ST2 cells were pretreated with rhAmbn or rhEnam at the indicated times, followed by stimulation with 1,25(OH)2D3 and DEX. Pretreatment with Ambn and Enam significantly reduced phosphorylation of p38 MAPK and JNK at 15 minutes. In addition, the phosphorylation of all MAPK was decreased at 30 minutes (Fig. 2B). Moreover, to address the effect of MAPK on RANKL expression, we incubated ST2 cells with MAPK inhibitors U0126 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), or JNK inhibitor II (JNK inhibitor) before treatment with 1,25(OH)2D3 and DEX. Both real-time RT8
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qPCR and western blot analysis demonstrated that U0126 and SB203580 suppressed RANKL expression induced with 1,25(OH)2D3 and DEX. In contrast, JNK inhibitor II enhanced the expression of RANKL insignificantly (Figs. 2C, D). Ambn and Enam inhibited osteoclast differentiation in co-culture system between ST2
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cells and BMCs To determine whether Ambn or Enam play a role in ST2 cell-supported osteoclast formation, ST2 cells were pretreated with rhAmbn or rhEnam prior to stimulation with
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1,25(OH)2D3 and DEX, and then co-cultured with BMCs. After 4 days, several TRAPpositive multinucleated osteoclasts were identified. We found that the differentiation of
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BMCs into multinucleated osteoclasts induced with 1,25(OH)2D3 and DEX was clearly decreased under the preincubation of ST2 cells in the presence of Ambn or Enam (Fig. 3). These results indicate that Ambn and Enam adversely affect osteoclastogenesis, suggesting that these proteins directly affect ST2 cells.
differentiation
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RANKL is an essential factor for multinucleated osteoclast formation and
To further examine the critical roles of RANKL on multinucleated osteoclast
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formation, we blocked ST2 cells with various concentrations of neutralizing antibody against RANKL (60–500 ng/ml), followed by induction with 1,25(OH)2D3 and DEX, and then co-
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culture with BMCs. The neutralizing antibody-treated groups obviously displayed lower numbers of multinucleated osteoclasts compared with mouse-IgG control groups, in a dosedependent manner (Fig. 4).
Discussion In the present study, we aimed to identify the role of Ambn and Enam on RANKL expression, and to verify the effects of Ambn and Enam on osteoclastogenesis. Our data revealed that exogenous Ambn and Enam suppressed RANKL expression, which attenuated 9
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multinucleated osteoclast formation. This finding is consistent with previous reports suggesting that Ambn-deficient mice exhibited lower bone volume density and higher trabecular bone separation in femurs [16,17]. Moreover, Ambn was found to promote bone mineralization by binding to CD63, and then inducing Src kinase inhibition in an
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osteosarcoma cell line [19]. In contrast, coating recombinant Ambn on cell culture plates increased the number of the multinucleated osteoclast formation by the integrin/ERK pathway [20], suggesting that Ambn has both direct and indirect effects on osteoclast
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formation. We do not have a specific explanation for these contrasting results, but it is possible that they reflect the differences in culture conditions, types of cells, concentrations
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of Ambn, and protocols used in the studies.
We also observed the enhancement of endogenous ERP gene expression in ST2 cells induced with 1,25(OH)2D3 and DEX, which was consistent with key time point marker genes of osteoblast differentiation (data not shown). However, the previous study demonstrated that commitment to osteoblast lineage was not mandatory for fibroblastic cell types to express
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RANKL, and mature osteoblasts are not a significant source of RANKL in bone [21]. Thus, in our study, ST2 cells serve as a supporting element for generating multinucleated
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osteoclasts from BMCs by producing RANKL with stimulation by 1,25(OH)2D3 and DEX. 1,25(OH)2D3 is one of the essential osteotropic factors that has been firmly reported to
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induce the differentiation of osteoclast progenitors cells in both direct and indirect pathways. 1,25(OH)2D3 itself has been found to promote multinucleated osteoclast formation [22]. Furthermore, 1,25(OH)2D3 indirectly supports osteoclastogenesis by stimulating the expression of RANKL in osteoblasts, and stromal cells pass through genomic and nongenomic pathways [23]. It has been found to activate RANKL gene transcription through vitamin D-responsive elements (VDRE) [24,25]. Additionally, DEX has been shown to enhance osteoclast formation through various mechanisms, including elevating VDR transcription and inducing the expression of M-CSF in ST2 and MC3T3E1 cells [26,27]. In 10
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this experiment, we could not detect the relevance of Ambn and Enam on nuclear translocation of VDR (Data not shown), but we found that Ambn and Enam decreased the phosphorylation of all MAPK induced with 1,25(OH)2D3 and DEX. However, only specific inhibitors against ERK1/2 and p38MAPK displayed an ability to contribute to RANKL
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expression in ST2 cells. MAPK proteins are known to play an essential role in controlling cell proliferation, differentiation, and gene expression. In mammals, MAPK can be divided into three main
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families: ERK1/2, JNK and p38 MAPK [28,29]. MAPK-specific inhibitors proved that activation of ERK and p38, specific MAPK proteins, was imperative for triggering RANKL
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expression in bone marrow stromal cells and osteoblasts [30]. Other studies have also supported the function of ERK1/2 and p38 in RANKL expression [30,31]. In our study, compared with p38, ERK1/2 has only minor effects on RANKL expression. From these results, the inhibitory effect of Ambn and Enam on the expression of RANKL induced with 1,25(OH)2D3 and DEX was found to depend primarily on the regulation of the p38 MAPK
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pathway.
In conclusion, our study demonstrated that Ambn and Enam might attenuate
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osteoclastogenesis by suppressing RANKL expression via MAPK signaling pathways in stromal cells. Further research is needed to precisely elucidate the molecular mechanism of
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ERPs on bone remodeling, which may impact therapies for bone metabolic diseases in the future.
Conflicts of Interest The authors have no financial conflicts of interest to disclose.
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Funding
This work was partially supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science [grant numbers 26463115]
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Factor in Murine Osteoblast-Like Cells, Endocr. 139 (1998) 1006-1012 [26] R. Kitazawa, K. Mori, A. Yamaguchi, T. Kondo, S. Kitazawa, Modulation of mouse RANKL gene expression by Runx2 and vitamin D3, J Cell Biochem. 105(2008) 1289-1297. [27] A.A. Hidalgo, K.K. Deeb, J.W. Pike, C.S. Johnson, D.L. Trump, Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription, J Biol Chem. 286 (2011) 36228-36237. [28] M. Cargnello, P.P. Roux, Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases, Microbiol. Mol. Biol. Rev. 75 (2011) 50-83 14
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[29] D. Morrison, MAP kinase pathways, Cold Spring Harb Perspect Biol, 2012, doi: 10.1101/cshperspect.a011254.
[30] Y. Mine, S. Makihira, Y. Yamaguchi, H. Tanaka, H. Nikawa, Involvement of ERK and p38 MAPK pathways on Interleukin-33-induced RANKL expression in osteoblastic cells,
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Cell Biol Int. 38 (2014) 655-662. [31] C. Rossa, K. Ehmann, M. Liu, C. Patil, K.L. Kirkwood, MKK3/ 6-p38 MAPK signaling is required for IL-1beta and TNF- alpha-induced RANKL expression in bone marrow stromal
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cells, J Interferon Cytokine Res 26(2006) 719–729.
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Figure Legends
Figure 1. Ambn and Enam suppressed RANKL expression on ST2 cells induced with 1,25(OH)2D3 and DEX. ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, then stimulated with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 24 hours
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(real-time RT-qPCR) or 36 hours (western blot analysis). (A) The mRNA level of RANKL and OPG was measured by real-time RT-qPCR. Data are expressed as the mean ± S.D. of triplicate cultures. *p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups. (B)
for RANKL. β-actin served as an internal control.
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Whole cell lysates were subjected to SDS-PAGE and western blot analysis with blot probes
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Figure 2. Ambn and Enam downregulated RANKL expression on ST2 cells via p38 MAPK and ERK1/2 signaling pathways. (A) ST2 cells were treated with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 15–180 minutes. (B) ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, prior to stimulation with 10-7 M
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1,25(OH)2D3 and 10-7 M DEX for 15 and 30 minutes. Whole cell lysates were subjected to western blot analysis for detecting MAPK expression and phosphorylation. (C, D) ST2 cells were blocked with 10 µM U0126, JNK inhibitor II or SB203580 for 1 hour followed by
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stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 24 (C) or 36 (D) hours. (C) The mRNA level of RANKL was measured by real-time RT-qPCR. Data are expressed as the
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mean ± S.D. of triplicate cultures. *p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups. (D) Whole cell lysates were subjected to western blot analysis with the blot probes for RANKL.
Figure 3. Ambn and Enam inhibit osteoclast differentiation in the co-culture system between ST2 cells and BMCs. (A) ST2 cells were pretreated with 100 ng/ml rhAmbn or 100 ng/ml rhEnam for 6 hours, prior to stimulation with 10-7 M 1,25(OH)2D3 and 10-7 M DEX. After 24 hours, ST2 cells were co-cultured with BMCs for 4 days. TRAP analysis was performed to detect multinucleated osteoclast formation. Scale bar 500 µm. (B) TRAP16
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positive cells containing more than three nuclei were counted. Data are expressed as the mean ± S.D. of triplicate cultures. *p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups. Figure 4. RANKL is essential for generating multinucleated osteoclasts. (A) ST2 cells were cultured in a medium containing 10-7 M 1,25(OH)2D3 and 10-7 M DEX for 22 hours,
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then blocked with 60–500 ng/ml anti-RANKL antibody or mouse IgG, followed by coculture with BMCs for 4 days. Scale bar 500 µm. (B) Several TRAP-positive multinucleated osteoclasts were counted. Data are expressed as the mean ± S.D. of triplicate cultures.
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*p<0.05, **p<0.01 versus 1,25(OH)2D3 and DEX-treated groups.
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ACCEPTED MANUSCRIPT Highlights •
Ambn and Enam affect RANKL expression through MAPK signaling pathway
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MAPK signaling pathway is necessary for RANKL expression in bone marrow stromal
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Ambn and Enam indirectly regulate multinucleated osteoclast formation.
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•
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cell.